U.S. patent number 5,741,715 [Application Number 08/452,742] was granted by the patent office on 1998-04-21 for quinidine immunoassay and reagents.
This patent grant is currently assigned to Roche Diagnostic Systems, Inc.. Invention is credited to Mitali Ghoshal, Kathryn Sarah Schwenzer, Robert Sundoro Wu.
United States Patent |
5,741,715 |
Ghoshal , et al. |
April 21, 1998 |
Quinidine immunoassay and reagents
Abstract
Novel quinidine derivatives are provided which can be used in an
improved immunoasssay for the detection of quinidine and quinidine
metabolites.
Inventors: |
Ghoshal; Mitali (Neshanic
Station, NJ), Schwenzer; Kathryn Sarah (Yardley, PA), Wu;
Robert Sundoro (West Orange, NJ) |
Assignee: |
Roche Diagnostic Systems, Inc.
(Branchburg, NJ)
|
Family
ID: |
23797741 |
Appl.
No.: |
08/452,742 |
Filed: |
May 30, 1995 |
Current U.S.
Class: |
436/537; 436/546;
530/388.9; 436/815; 530/389.8; 530/807 |
Current CPC
Class: |
C07D
453/04 (20130101); G01N 33/9453 (20130101); Y10S
436/815 (20130101); Y10S 530/807 (20130101) |
Current International
Class: |
C07D
519/00 (20060101); C07D 453/04 (20060101); C07D
453/00 (20060101); G01N 33/94 (20060101); G01N
033/542 (); G01N 033/577 (); G01N 033/533 (); C07K
016/44 () |
Field of
Search: |
;546/177,178
;530/389.8,807,388.9 ;436/815,822,546,537 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
|
|
|
087564 |
|
Jul 1983 |
|
EP |
|
A-O 254 120 |
|
Jan 1988 |
|
EP |
|
A-O 279 213 |
|
Aug 1988 |
|
EP |
|
85/00605 |
|
Feb 1985 |
|
WO |
|
WO-A-94 24559 |
|
Oct 1994 |
|
WO |
|
WO-A-95 03296 |
|
Feb 1995 |
|
WO |
|
Other References
M Wolff et al., Med. Chem. Res., vol. 1, pp. 101-108 (1991). .
A. Soto et al., Clinical Chemistry, vol. 29, No. 6, pp. 1200-1201
(1983). .
C. Regan, J. Pharm. Pharmacol., vol. 38, pp. 834-836 (1986). .
A. Sidki et al., Cliin. Chem., vol. 33, No. 4, pp. 463-467 (1987).
.
Mattingly, P.G., Bioconjugate Chemistry, 3:430-431 (1992)..
|
Primary Examiner: Ceperley; Mary E.
Attorney, Agent or Firm: Johnston; George W.
Rocha-Tramaloni; Patricia S. Semionow; Raina
Claims
We claim:
1. A kit for performing a fluorescence polarization immunoassay to
determine the concentration of quinidine in body fluid samples,
said kit comprising the tracer
(9S)-N-[3',6'-dihydroxy-3-oxospiro[isobenzofuran-1(3H),9'-[9H]xanthen]5-yl
)methyl]-4-[(9-hydroxycinchonan-6'yl)oxy]butamide and antibody MoAb
Q6-6C5 prepared by inoculating a host animal with an immunogen of
formula ##STR11## wherein Z is bovine thyroglobulin (BTG), said
antibody having a dynamic curve span of at least 150 mP and having
a cross reactivity not exceeding the given percentages to the
following quinidine metabolite compounds: quinidine-N-oxide 13.5%;
3-S-hydroxyquinidine 11%; 2-oxoquinidine 3%; and
O-desmethylquinidine 43.5%.
2. Antibody MoAbQ6-6C5 prepared by inoculating a host animal with
the compound of the formula ##STR12## wherein Z is bovine
thyroglobulin (BTG), said antibody having a dynamic curve span of
at least 150 mP and having a cross reactivity not exceeding the
given percentages to the following quinidine metabolite compounds:
quinidine-N-oxide 13.5%; 3-S-hydroxyquinidine 11%; 2-oxoquinidine
3%; and O-desmethylquinidine 43.5%.
Description
BACKGROUND OF THE INVENTION
The present invention relates to reagents used for the quantitative
determination of quinidine in serum. In particular, the present
invention relates to an improved fluorescence polarization
immunoassay utilizing novel quinidine derivatives, as well as novel
haptens, antibodies and tracers produced from said novel
derivatives, as reagents in such assays.
Quinidine is a pharmaceutical agent generally prescribed for
regulation of arrythmic heartbeat and thus its concentration in a
patient's blood is critical and is carefully monitored during its
administration. Serum quinidine levels of 1.5 to 5 mg/mL have been
reported as therapeutic, based on nonspecific methodologies that
measure quinidine metabolites as well as quinidine (Physician Desk
Reference. 46th ed. Montvale, N.J.; Medical Economic Data;
1993:688-689). Quinidine was the first anti-arrhythmic for which
the efficacy of therapeutic monitoring was demonstrated. The
therapeutic concentration range for quinidine is quite narrow, and
toxic effects due to overdosage can mimic symptoms of heart
disease. The dosage required to achieve therapeutic serum levels is
dependent on the drug formulation, patient age, severity and nature
of the cardiac disorder and on individual variability in drug
absorption and metabolism. Thus, monitoring of serum quinidine
levels provides direct evidence to guide the physician in
determining drug dosage for each individual patient.
The level of quinidine in serum samples can be determined through
competitive binding immunoassays. Competitive binding immunoassays
for measuring the concentration of an analyte (also referred to as
a ligand) such as the drug quinidine, in a test sample are based on
the competition between a ligand in a test sample and a labeled
reagent, referred to as a tracer, for a limited number of receptor
binding sites on antibodies specific to the ligand and tracer. The
concentration of ligand in the sample determines the amount of
tracer that will specifically bind to an antibody. The amount of
tracer-antibody conjugate produced may be quantitatively measured
and is inversely proportional to the quantity of ligand in the test
sample.
Fluorescence polarization (FP) provides a quantitative means for
measuring the amount of tracer-antibody conjugate produced in a
competitive binding immunoassay. In general, fluorescent
polarization techniques are based on the principle that a
fluorescence labeled compound when excited by linearly polarized
light will emit fluorescence having a degree of polarization
inversely related to its rate of rotation. When a molecule, such as
a tracer-antibody conjugate having a fluorescent label is excited
with linearly polarized light, the emitted light remains highly
polarized because the fluorophore is constrained from rotating
between the time light is absorbed and emitted. When a "free"
tracer compound (i.e. unbound to an antibody) is excited by
linearly polarized light, its rotation is much faster than the
corresponding tracer-antibody conjugate and the molecules are more
randomly oriented, therefore the emitted light is depolarized.
In fluorescence polarization immunoassays (FPIA), fluorescence
polarization is a reproducible function of the ligand or drug
concentration, and thus is suitable for the quantitative
determination of drug concentrations in serum for the purpose of
therapeutic drug monitoring. When tracer, serum containing
antibodies specific for the drug to be measured (for example,
quinidine) and drug-free patient serum are mixed together, most of
the tracer binds to the antibodies. As a result, when the bound
tracer is excited with polarized light at 489 nm, the light emitted
at 520 nm remains highly polarized. However, if drug is present in
the patient sample, the drug will compete with the tracer for
binding to the antibodies. Thus, more of the tracer will remain
unbound and the emitted light is depolarized.
An FPIA according to the present invention can be any type of
automated or manual FPIA. Preferably the FPIA is carried out on the
automated COBAS FARA II.RTM. chemistry system (COBAS FP assay
system, Roche Diagnostics, Inc., Somerville, N.J.) to measure the
binding of fluorescein labeled drug (tracer) to specific antibodies
(see Dandliker and Feigen, Biochem. Biophys. Res. Comm. 5: 299,
1961).
The COBAS FP assay system measures the fluorescence polarization
resulting from the interaction of fluorescein labelled tracer,
antibody and calibrators containing known amounts of drug, such as
quinidine, in human serum. From the measurements a curve relating
drug concentration to millipolarization (mP) units is produced. The
precision of drug concentration measurement is related to dynamic
span of the standard curve and relative intensity of the tracer in
solution. When a maximum amount of tracer is bound to the antibody
in the absence of drug in the serum, maximum polarization in mP
units is measured. "Span" (also known as "dynamic curve span")
indicates the difference between the minimum and maximum
millipolarization units produced by tracer bound to antibody. A
larger span indicates better precision and sensitivity of tracer
performance. "Intensity" is a measure of the strength of the
fluorescence signal above the intensity of the background
fluorescence. Intensity of a tracer preferably remains constant
throughout the life of the reagent. Free tracer depolarizes light
yielding 20-75 mP. A good dynamic span ranges from 150-250 mP.
Subsequently, the tracer, antibody and patient's serum are allowed
to interact under the same conditions which generated the
calibration curve. The mP units thus obtained can be correlated
accurately to the drug level in the patient's serum by comparison
with the calibration curve in the assay.
Fluorescein-labeled quinidine compounds are known, for example
5-aminofluorescein-labeled quinidine (see U.S. Pat. No. 4,585,862),
DTAF-labeled quinidine (see U.S. Pat. No. 4,420,568),
.beta.-galactosyl-umbelliferone-labeled quinidine (see EP
83100413.0) and enzyme labeled quinidine (see WO 85/00605).
However, some of these compounds contain types of linkages that are
susceptible to hydrolysis, therefore shortening the shelf life of
the tracers, for example carbamate ester and O-triazinyl ether
linkages in quinidine tracers derived out of the C-9 position of
quinidine (see U.S. Pat. Nos. 4,420,568 and 4,585,862).
A method to prepare quinidine derivatives out of the C-6 position
on the quinidine molecule has been described for making an enzyme
labeled-quinidine using N-succinimidyl 3-(2-pyriyldithio)propionate
(SPDP) (see WO 85/00605), but not for making fluorescein-labeled
quinidine tracers. This method produces a dialkylkated product out
of the C-6 position in the quiniclidine molecule which results in
an unstable tracer due to the positive charge at the quiniclidine
ring prone to hydrolytic cleavage.
Therefore, it is an object of the present invention to provide a
stable quinidine derivative substituted exclusively at the 6
position of the quinidine molecule.
It is a further object of the present invention to provide a stable
fluorescein tracers derived from 6-substituted quinidine
derivatives having an amide linkage between the fluorescein
molecule and the quinidine derivative.
An additional object of the present invention is to provide
reagents, such as antibodies, derived form the quinidine
derivatives for an improved fluorescence immunoassay to quantitate
quinidine in body fluid samples.
SUMMARY OF THE INVENTION
The present invention relates to novel quinidine derivatives of the
formula ##STR1## wherein L is a linking group consisting of from 1
to 10 carbon atoms, which may be straight or branched chain, and
may be saturated or unsaturated, and may include from 0-3
heteroatoms. F is a functional group selected from the groups
consisting of amino, carboxyl, sulhydryl, imino, and maleimide.
The compounds of formula I are useful as haptens in the preparation
of immunogens as well as useful to prepare labelled quinidine
tracers.
The invention also relates to antibodies produced against the novel
quinidine derivatives of the present invention. The invention
further relates to a kit containing the reagents of the present
invention for performing an improved fluorescence polarization
immunoassay.
BRIEF DESCRIPTION OF THE FIGURES
The present invention may be more readily understood by reference
to the following figures. The numbers following the compounds
correlate to numbers shown in the figures.
FIG. 1 shows the formulae of the starting materials and
intermediates involved in the synthesis of
(9S)-4-[(9-hydroxycinchonan-6'yl)oxy]-1-iminobutyl-[BTG] (4);
FIG. 2 shows the formulae of the starting materials and
intermediates involved in the synthesis of
(9S)-N-[(3',6'-Dihydroxy-3-oxospiro[isobenzofuran-1(3H),9'-[9H]xanthen]-5y
l) methyl]-4-[(9-hydroxycinchonan-6'-yl)oxy]butamide (7);
FIG. 3 shows the formulae of the starting materials and
intermediates involved in the synthesis of
(9S)-[2-[[(3',6'-Dihydroxy-3-oxospiro[isobenzofuran-1(3H),9'-[9H]pantheon]
-5-yl) methylamino]-2-oxoethyl]carbamic acid
6'-methoxycinchonan-9-yl ester (10);
FIG. 4 shows the formulae and starting materials involved in the
synthesis of (9S)-3',6'-Dihydroxy-N-[3-(9-hydroxycinchonan-6'-yl)
oxy]propyl]-3-oxospiro[isobenzofuran-1 (3H),
9'-[9H]xanthene]-5-carboxamide (13);
FIG. 5 shows comparison testing of monoclonal antibody MoAb Q6-6C5
and polyclonal antibody 796 binding to the tracer
(9S)-N-[(3',6'-Dihydroxy-3-oxospiro[isobenzofuran-1(3H),9'-[9H]xanthen]-5y
l) methyl]-4-[(9-hydroxycinchonan-6'yl)oxy]butamide (7);
FIG. 6 shows the comparison testing of monoclonal antibody MoAb
Q6-6C5 with the tracer
(2(S)-exo,syn)-8-ethenyl-1-(2-((3',6'-dihydroxy-3-oxospiro(isobenzofuran-1
(3H),9'-(9H)xanthen-5-yl)amino)-2-oxoethyl)-2-(hydroxy(6-(2-ethoxy-2-oxoeth
oxy) -4-quinolineyl)methyl)-1-azoniabicyclo(2.2.2)octane chloride
hydrochloride (14), and the tracer
(9S)-N-[(3',6'-Dihydroxy-3-oxospiro[isobenzofuran-1(3H),9'-[9H]xanthen]-5y
l) methyl]-4-[(9-hydroxycinchonan-6'-yl)oxy]butamide (7)
formulations;
FIG. 7 shows the stability of the tracer
(9S)-N-[(3',6'-dihydroxy-3-oxospiro[isobenzofuran-1(3H),9'-[9H]xanthen]-5y
l) methyl]-4-[(9-hydroxycinchonan-6'-yl)oxy]butamide (7) incubated
at 45.degree. C.;
FIG. 8 shows the stability of the tracer
(9S)-N-[(3',6'-dihydroxy-3-oxospiro[isobenzofuran-1(3H),9'-[9H]xanthen]-5y
l) methyl]-4-[(9-hydroxycinchonan-6'-yl)oxy]butamide (7) incubated
at 37.degree. C.; and
FIG. 9 shows the comparison of the dynamic span in an FPIA using
the novel tracer
(9S)-N-[(3',6'-dihydroxy-3-oxospiro[isobenzofuran-1(3H),9'-[9H]xanthen]-5y
l) methyl]-4-[(9-hydroxycinchonan-6'-yl)oxy]butamide (7) and MoAb
Q6-6C5 and a commercially available reagent system.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to novel quinidine derivatives of the
formula ##STR2## wherein L is a linking group consisting of from 1
to 10 carbon atoms, which may be straight or branched chain, and
may be saturated or unsaturated, and may include one or more
aromatic groups and may include 0-3 heteroatoms. Linking groups are
well known to those of skill in the art (see e.g. U.S. Pat. No.
4,160,016). F is a functional group selected from amino, carboxy,
sulfhydryl, imino, and maleimide. The meaning of the term
"functional groups" is apparent to those of skill in the art.
Preferably L is from 1-5 carbon atoms. Preferably the carbon atoms
are unsaturated and the unsaturated group includes an aromatic
group such as phenyl. Preferably the heteratoms include O, N, and
S. F is most preferably carboxyl or amino.
The novel quinidine derivatives of the present invention are used
for preparing antibodies to quinidine and quinidine derivatives, as
well as for bonding to detector molecules to form tracers for use
in fluorescent polarization assays for the detection of quinidine
in body fluid samples.
The present invention provides a quinidine derived hapten. A hapten
is a compound capable of eliciting an immune response in a
challenged animal in order to generate antibodies against the
compound, e.g. a quinidine derivative, for use in an FPIA. An
antibody used in the quinidine FPIA preferably is specific for
quinidine and will not react with quinidine-like compounds such as
quinine (shown below). The hapten used to prepare the preferred
immunogen of the present invention was derivatized at the C-6'
position on the quinidine molecule. ##STR3##
An alkyl group is attached at the C-6' position of quinidine to
introduce a spacer. The preferred amount of alkylating reagent used
to prepare the hapten and other quinidine derivatives of the
present invention is one molar equivalent, the most preferred
amount being less than one molar equivalent.
The hapten is used to prepare an immunogen- a conjugate of the
hapten and a protein carrier molecule- to elicit an antibody
response. An immunogenic carrier molecule is a macromolecule
capable of independently eliciting an immunological response in a
host animal and which can be coupled to a quinidine derivative of
the present invention. Suitable immunogenic carrier molecules are
protein carriers which include bovine serum albumin (BSA), keyhole
limphet hemocyanin (KLH) polypeptides and bovine thyroglobulin
(BTG).
The structure of the immunogen of the present invention is as
follows: ##STR4## wherein L is a linking group consisting of from 1
to 10 straight or branched chain, saturated or unsaturated carbon
atoms; preferably of from 1-5 carbon atoms. Preferably the carbons
are unsaturated and include at least one aromatic group such as
phenyl. L may also include 0-3 heteroatoms selected from O, N and
S. F is a functional group selected from amino, carboxyl,
sulfhydryl, imino, and maleimide. F is most preferably carboxyl or
amino. Y is an immunogenic carrier molecule such as a protein; a
polysaccharide such as dextran or an oligosugar; or a naturally
occuring or synthetic polyaminocarboxylic acid such as polylysine
or polyglycine.
A preferred immunogen of the present invention is illustrated by
the formula ##STR5## wherein Z is a protein carrier. Z can include
albumin, bovine serum albumin (BSA), key-hole limpet hemocyanin
(KLH), bovine thyroglobulin (BTG), egg ovalbumin, bovine gamma
globulin, small natural polypeptides such as gramicidin, and
various synthetic polypeptides. The preferred immunogen of the
present invention contains an imino as the functional group F and
BTG as the protein carrier Z. A preferred host animal for
production of the antibody includes mice.
The preferred quinidine immunogen is prepared from a hapten bearing
a mono-substituted imidate ester wherein L is 3 carbon atoms. The
process for making the quinidine immunogen of the present invention
is shown in FIG. 1 and detailed in examples 1,2,3 and 7. The first
step is the demethylation of quinidine to yield 6-hydroxycinchonine
(1) according to known methods (see L. D. Small, et al., J. Med.
Chem. vol. 22, 1014-1016, 1979). The second step is alkylation of
6-hydroxyquinidine(1) to
(9S)-4-[(9-Hydroxycinchonan-6'yl)oxy]butanenitrile (2). Alkylating
agents that can be used include 4-bromobutyronitrile,
chloroacetonitrile, 3-chloropropionitrile, and 3-(bromomethyl)
benzonitrile. The preparation of derivatives wherein L is less than
4 is given in Examples 4, 5 and 12; derivatives wherein L contains
phenyl in Example 6.
The preferred amount of alkylating reagent used is one molar
equivalent of, for example 4-bromobutyronitrile. Most preferably
used is 0.9 molar equivalent of alkylating reagent. Compound (2) is
the preferred compound of the present invention used to generate
the preferred monoclonal antibody Q6-6C5 (MoAb Q6-6C5).
In contrast, the use of more than one molar equivalent of
alkylating reagent results in disubstituted quinidine derivatives,
shown below. ##STR6## The syntheses of the above undesirable
compounds,
(S)-8-Ethenyl-2-[hydroxy-6-(2-ethoxy-2-oxoethoxy)-4-quinolinylmethyl]-1-(2
-ethoxy-2-oxoethyl)azoniabicyclo[2.2.2]octane iodide (15) and
(9S)-1-(3-carboxypropyl)-6'-(3-carboxypropoxy) cinchoninum chloride
(16) using two molar equivalents of alkylating reagent are
described in Examples 9 and 10.
The third step of the immunogen synthesis is converting the nitrile
on the hapten to an imidate ester for subsequent coupling of the
hapten to a protein. The use of imidate ester containing molecules
for coupling to proteins' is known, for example, using a
cross-linking reagent such as dimethylsuberimidate dihydrochloride
(Pierce). The use of this cross-linker, however, can result in
polymerization of the proteins causing the precipitation of the
hapten conjugate. Also known is the reaction of an imidate ester
with an amine to form amidine. It is preferable, however, in the
present invention to have a monofunctionalized active group, i.e.
an imidate ester, directly on the hapten for the most effective
protein coupling.
A monofunctionalized imidate ester derivative of cocaine has been
used to make a cocaine immunogen (see U.S. Pat. No. 4,045,420).
Sodium methoxide in methanol was used to convert the nitrile to
benzoyl ecgonine bearing an imidate ester for protein coupling.
This method, however, is not suitable for preparing the quinidine
immunogen of the present invention due to the presence of the C-9
hydroxy group on the molecule which is capable of crossreacting
with the imidate ester as soon as it is formed.
A preferred method of making the immunogen of the present
invention, therefore, is through an acid catalyzed reaction. The
imidate ester is generated by treatment of the nitrile with HCl in
methanol. The resulting imidate ester forms a hydrochloride salt
with amino groups on the molecule. The imidate ester is preferably
used for protein coupling soon after it is made. In the preferred
embodiment compound (2) is treated with hydrochloric acid gas in
methanol to form (9S)-4-[(9-hydroxycinchonan-6'-yl)butanimidic acid
methyl ester (3).
Alternate methods to prepare the quinidine hapten include forming a
hapten conjugate wherein the functional group F is carboxy.
Typically used is the direct coupling of a carboxyl group to an
amine using carbodiimide. An alkylating agent is attached to the
hydroxyl group of cinchonine (see example 12). The carboxy end is
activated at the hydroxyl using DCC (dicyclohexyl carbodiimide) and
NHS (N-hydroxysuccinimide) to form the active ester. The active
group couples to a protein carrier under relatively mild reaction
conditions (see U.S. Pat. Nos. 5,101,015 and 4,329,281).
Also widely used are sulfhydryl reactive maleimide- or
.alpha.-haloacetamide-linker, known to react selectively with
thiols to form thioether-linked conjugates. A hapten conjugate
wherein the functional group F is a maleimido group can be linked
to proteins bearing sulhydryl groups (see example 18). The amine
reacts with the maleimido derivative for effective coupling to
another molecule bearing sulfhydryl residues. Maleimido-NHS active
ester compounds are commercially available as bifunctional linkers,
such as succinimidyl 4-(p-maleimidomethyl)cyclohexane-1-carboxylate
(SMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),
succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), and derivatives
thereof.
Bifunctional linkers, such as SPDP, may also be used to link the
hapten and protein together to form a disulfide-containing
conjugate. However, the preparation of either the thioether-linked
or the disulfide-linked conjugates is a multistep procedure,
whereas the coupling of carboxyl groups to proteins is a more
direct procedure.
The quinidine immunogens of the present invention were used to
generate polyclonal and monoclonal antibodies, for example the
novel monoclonal antibody MoAb Q6-6C5 and polyclonal antibody P796.
The use of the novel monoclonal antibody in conjunction with the
novel tracers of the present invention (described below), results
in an improved immunoassay for the detection and quantitation of
quinidine.
For the preferred antibody of the present invention cross
reactivity should not exceed, for the following quinidine
metabolite compounds: quinidine-N-Oxide 13.5%: 3-S-hydroxyquinidine
11%; 2'-oxoquinidine 3%: O-desmethylquinidine 43.5%. The preferred
monoclonal Ab also has dynamic curve span of at least 150 mP.
The antibodies were tested to determine their efficiency in binding
a new tracer of the present invention. FIG. 5 shows the comparison
between using the polyclonal antibody and the monoclonal antibody
with the tracer of the present invention in the FPIA. Both
antibodies demonstrate binding to the tracer. The curves indicate
that the monoclonal antibody has a larger dynamic span and
therefore increased sensitivity in the FPIA.
Table 1 shows the cross-reactivity of MoAb Q6-6C5 towards various
metabolites of quinidine including quinine as determined in the
FPIA. Cross reactivity with quinine is shown to be insignificant,
therefore quinine will not interfere with the performance of the
quinidine assay. MoAb Q6-6C5 demonstrates lower cross-reactivity to
the metabolites (with the exception of dihydroquinidine) thus
providing a more precise and highly specific method for monitoring
quinidine in serum. These results were obtained using an assay
protocol such as the one described in Example 22.
TABLE 1
__________________________________________________________________________
CROSS REACTIVITY New Kit Current Kit using using Concentration
MoAbQ6-6C5 & Tracer of Test Tracer 7 14 TDx Test Compound %
Cross- % Cross- % Cross- Compound Added (ug/mL) Reactivity
Reactivity Reactivity
__________________________________________________________________________
Quinidine-N-Oxide 10 13.5 30.5 31.9 100 4.9 H 7.2
3-S-Hydroxyquinidine 10 11 21 18.8 100 3.7 4.3 5.3 2'-Oxoquinidine
10 3 20 3.3 100 1 3.7 1.1 500 0.53 1.55 0.58 1000 0.35 H 0.39
O-Desmethylquinidine 1 55 55 206 10 43.5 46.5 60.3 Dihydroquinidine
10 H 67 79 5 160 76 88 2.5 156 76 96 10,11-Dihydro 10 1 14.5 3.9
quinidine-diol 100 1.7 3.2 1.8 Quinine 10 <0.1 not tested 100
<0.1 <0.1 0.8 1000 <0.1 not tested
__________________________________________________________________________
H: High
The present invention also provides novel quinidine-derived
fluorescein labelled tracers for use in fluorescent polarization
immunoassays. Fluorescein compounds are known in the art (Molecular
Probes Publication, Eugene, Oreg.; Bioconjugate Chemistry, 1192, 3,
430-431, 1992; U.S. Pat. No. 4,668,640) and have been utilized to
make fluorescein-labeled quinidines, for example
5-aminofluorescein-labeled quinidine (see U.S. Pat. No. 4,585,862),
DTAF-labeled quinidine (see U.S. Pat. No. 4,420,568),
.beta.-galactosylumbelliferone-labeled quinidine (see EP
83100413.0) and enzyme labeled quinidine (see WO 85/00605).
Known fluorescein-labeled quinidines are derived from the C-9
position of the quinidine molecule and contain linker arms of
carbamate ester and O-triazinyl ether (see U.S. Pat. Nos. 4,420,568
and 4,585,862). However, these types of known linkages are
susceptible to hydrolysis and therefore not suitable for making the
fluorescein tracer of the present invention.
Another commercially available fluorescein labelled tracer,
((3',6'-dihydroxy-3-oxospiro(isobenzofuran-1(3H),9'-(9H)xanthen-5-yl)amino
)-2-oxoethyl)-2-(hydroxy(6-(2-ethoxy-2-oxoethoxy)-4-quinolineyl)methyl)-1-a
zoniabicyclo(2.2.2)octane chloride hydrochloride (14), bears a
positive charge on the quinuclidine ring (shown below), causing
instability of the compound. ##STR7##
The structure of the fluorescence polarization tracer of the
present invention is represented by the formula ##STR8## wherein L
and F are as previously defined. X is a detector molecule selected
from the group consisting of a chemiluminescent, such as luciferin,
umbelliferone and napthalene-1,2-dicarboxylic acidhydrazide (U.S.
Pat. No. 4,331,808); an energy donor molecule, such as a
fluorescein "Q" as defined below or Texas Red; and a radiolabelled
group such as I.sup.125 -tyramine, C.sup.14 labelled compounds,
such as methyl iodide or amino acids.
The preferred tracer of the present invention has the formula
##STR9## wherein F is selected from amino or carboxy and Q is a
fluorescence emitting compound such as 5-carboxyfluorescein,
6-carboxyfluorescein, 5-aminofluorescein, 6-aminofluorescein,
5-fluorescein isothiocyanate, 6-fluorescein isothiocyanate,
4'-aminomethylfluorescein, 5-aminomethylfluorescein and derivatives
of aminofluorescein, e.g. glycinated fluorescein. Most preferred
are aminomethyl fluorescein and carboxyfluorescein.
The group Q may be selected from a variety of commercially
available detector molecules (Molecular Probes, Inc. Eugene, Oreg.)
and attached to the quinidine derivatives by methods known in the
art to provide reagents useful in different assay formats. In
addition to fluorescein, detector molecules such as radiolabels or
chemiluminescent detector molecules, for example, tyramine can also
be used.
FIG. 2 illustrates the method for preparing the most preferred
novel FP tracer of the present invention
(9S)-N-[(3',6'-Dihydroxy-3-oxospiro
[isobenzofuran-1(3H),9'-[9H]xanthen]-5yl)
methyl]-4-[(9-hydroxycinchonan-6'-yl)oxy]butamide (7). The
synthesis of this tracer is described in examples 11-13.
The first step in the synthesis of the tracer of the present
invention, as for the hapten, is the alkylation of
6-hydroxyquinidine using preferably one or less molar equivalent of
alkylating reagent. The first step is the alkylation of
6-hydroxyquinidine(1) to (9S)-4-[(9-Hydroxycinchonan-6'-yl)
oxybutanoic acid ethyl ester (5) using one molar equivalent of
ethyl 4-bromobutyrate, most preferably using 0.9 molar equivalent
of ethyl 4-bromobutyrate. The alkylated product (5) is hydrolyzed
to obtain the corresponding acid
(9S)-4-[9-Hydroxycinchonan-6'-yl)oxybutanoic acid (6). The acid is
activated to form N-hydroxysuccinimide using a method well-known in
the literature. This activated ester is coupled with
5-aminomethylfluorescein in the presence of pyridine to give the
quinidine tracer
(9S)-N-[(3',6'-dihydroxy-3-oxospiro[isobenzofuran-1(3H),9'-[9H]xanthen]-5y
l) methyl]-4-[(9-hydroxycinchonan-6'-yl)oxy]butamide (7).
The linkage between the antigenic moiety and the fluorescent moiety
in the tracer can be an amidine linkage as in the novel hapten
structure. However, the linkage between the antigenic quinidine
moiety and the fluorescein molecule in the tracer is preferably
different from the amidine linkage used to prepare the immunogen.
Linkages to be used to form the tracer can include amide, urea,
thiourea, amidine, ether, and thioether. The preferred linkage for
the tracer in the present invention is an amide bond which provides
more stability to the tracer.
The new tracer (9S)-N-[(3',6'-dihydroxy-3-oxospiro
[isobenzofuran-1(3H),9'-[9H]xanthen]-5yl)
methyl]-4-[(9-hydroxycinchonan-6'-yl)oxy]butamide (7) bears a
neutral charge on the molecule, in contrast, tracer (14) has a
positive charge on the quinuclidine ring. The new tracer is highly
stable at 37.degree. C. as shown in FIG. 8, an important property
for the performance of the tracer in the FPIA assay. An accelerated
stability study conducted at 45.degree. C. for 4 weeks demonstrated
no loss of curve span (see FIG. 7). The C-6 tracer also performed
well with MoAb Q6-6C5. The tracer-antibody pair exhibited a large
dynamic curve span (204 mP) demonstrating a better precision and
sensitivity in the measurements of analytes over a known tracer
compound (14) (151 mP), as shown in FIG. 6.
FIG. 8 illustrates the comparison in the performance in the FPIA
between the new C-6 tracer (7) and MoAb Q6-6C5 pair of the present
invention and a tracer and antibody pair from a commercially
available FPIA kit for quinidine (TDx, Abbott Laboratories). The
standard curve shown in FIG. 8 indicates that the tracer of the
present invention (7) has a larger dynamic span than that obtained
with the TDx assay, therefore demonstrating better sensitivity and
precision in measurement of quinidine.
Tracers synthesized out of an alternate position on the quinidine
molecule, for example the C-9 position can be prepared. FIG. 3
illustrates the method of preparing the C-9 tracer
(9S)-[2-[[(3',6'-Dihydroxy-3
-oxospiro[isobenzofuran-1(3H),9'-[9H]pantheon]-5-yl)
methylamino]-2-oxoethyl]carbamic acid 6'-methoxycinchonan-9-yl
ester (10). Quinidine is treated with ethylisocyanatoacetate at
room temperature to give the carbamate ester (8). The ester (8) is
hydrolyzed to provide the corresponding acid (9). The acid (9) is
converted to the active ester by reaction with dicyclohexyl
carbodiimide and N-hydroxysuccinimide followed by coupling with
5-aminomethyl fluorescein in pyridine to give the C-9 quinidine
tracer (10). The synthesis is further detailed in Examples
14-16.
However, when tested in the FP immunoassay, the C-9 (10) tracer did
not bind to the preferred quinidine antibody MoAb Q6-6C5. Table 2
demonstrates the binding ability of the monoclonal antibody MoAb
Q6-6C5 to the C-6 and C-9 tracers. The ability of binding is
expressed as the degree to which polarization is retained by the
antigen-antibody complex as indicated in mP units. As Table II
below indicates, increasing the concentration of MoAb Q6-6C5 to
1:20 titer had no effect on the C-9 tracer, hence no binding of
tracer to the antibody, and no standard curve could be generated.
In comparison, the tracer derived out of the C-6 position showed
good binding to the monoclonal antibody and a high degree of
polarization that is retained.
TABLE 2 ______________________________________ COMPARISON C-6 (7)
TRACER vs C-9 (10) FP TRACER ON MOAB Q6-6C5 MoAb Q6-6C5, titer (C-9
tracer) (C-6 tracer) ______________________________________ 1:20
28.6 mP 1:40 25.3 mP 1:525 -- 240 mP 1:550 -- 238 mP
______________________________________
As demonstrated by the results in Table 2, it is important to the
performance of the FPIA to have both the antibody and tracer
produced from compounds which were derived from the C-6 position on
the quinidine molecule.
Fluorescein molecules other than aminomethylfluorescein can be used
to prepare the novel quinidine fluorescence polarization tracers of
the present invention. The use of 5-carboxyfluorescein has been
shown in other FPIAs (see U.S. Pat. No. 4,668,640). However,
extensive modification of quinidine is required to introduce an
amino linker arm for effectively linking the quinidine to the
carboxyfluorescein. Methodology to make a carboxyfluorescein
labeled quinidine derivative out of the C-6 position has not been
published.
A preferred carboxy-fluoroscein tracer of the present invention has
the formula ##STR10##
FIG. 4 illustrates the general method for preparing the
(9S)-3',6'-Dihydroxy-N-[3-[(9-hydroxycinchonan-6'-yl)
oxy]propyl]-3-oxospiro[isobenzofuran-1 (3H),
9'-[9H]xanthene]-5-carboxamide (13) and Examples 17-19 provide the
synthesis of the compound.
The carboxyfluorescein tracer (13) used in the FPIA with the
polyclonal antibody P796 derived from the same quinidine immunogen
used to prepare MoAb Q6-6C5 demonstrated a good dynamic span (e.g.
>150 mP). The data shown in Table 3 demonstrate that the
carboxyfluorescein tracer is stable at 37.degree. C. and performs
well in the assay.
TABLE 3 ______________________________________ Quinidine polyclonal
antibody and carboxy tracer (13) stability (mP units). 37.degree.
C. 37.degree. C. 37.degree. C. ug/mL Day 0 1 week 4 week 6 week
______________________________________ 0 239.4 236.9 234.9 232.4
0.5 229.8 214.7 207.8 200.7 1 198 181.2 171 168.9 2 131.4 119.8
115.5 118.2 4 95.6 93 94.3 94.5 8 77.3 77.1 78.5 79.4
______________________________________
The novels tracers and antibody of the present invention can be
provided in an immunoassay kit for the determination of quinidine
amounts in sample body fluids such as serum. In a preferred
embodiment the kit will be used for performing the immunoassay on
the automated COBAS FARA II.RTM. chemistry system (COBAS FP assay
system, Roche Diagnostics, Inc., Somerville, N.J.) A kit for
performing a flourescence polarization immunoassay to determine the
concentration of quinidine in human serum comprises a tracer
compound of formula IV, for example compound 7 or 13, and an
antibody, for example MoAb Q6-6C5, generated from a compound of
formula II.
EXAMPLES
The following are non-limiting examples which illustrate the
synthesis of the novel quinidine derivatives of the present
invention and the use of these compounds in a fluorescence
polarization immunoassay system. The numerical designations of the
compounds in the headings and in Examples 1-19 refer to the
structural formulae shown in FIGS. 1 through 4.
The chemical structures of the intermediates and final product of
the synthesis of
(9S)-4-[(9-hydroxycinchonan-6'yl)oxy]-1-iminobutyl-[BTG] (4) are
shown in FIG. 1. The chemical structures of the intermediates and
final product of the synthesis of
(9S)-N-[(3',6'-dihydroxy-3-oxospiro[isobenzofuran-1(3H),9'-[9H]xanthen]-5y
l) methyl]-4-[(9-hydroxycinchonan-6'-yl)oxy]butamide (7) are shown
in FIG. 2. The chemical structures of the intermediates and final
product of the synthesis of
(9S)-[2-[[(3',6'-Dihydroxy-3-oxospiro[isobenzofuran-1(3H),9'-[9H]pantheon]
-5-yl) methylamino]-2-oxoethyl]carbamic acid
6'-methoxycinchonan-9-yl ester (10) are shown in FIG. 3. The
chemical structures of the intermediates and final product of the
synthesis of (9S)-3',6'-Dihydroxy-N-[3-[(9-hydroxycinchonan-6'-yl)
oxy]propyl]-3-oxospiro[isobenzofuran-1 (3H),
9'-[9H]xanthene]-5-carboxamide (13) are shown in FIG. 4.
Example 1
Preparation of 6-Hydroxycinchonine(1).
A 11 three-necked, round-bottom flask equipped with a condenser
under argon atmosphere was charged with 5.0 g (15.4 mmol) of
quinidine (97%, Aldrich) and 500 ml of dichloromethane. The
solution in the flask was cooled to -78.degree. C. To this cooled
solution was slowly added 61 ml (61 mmol) of 1.0M borontribromide
in dichloromethane over a period of 45 minutes. The reaction
mixture was allowed to warm up to room temperature for a period of
2.5 h and was heated to reflux for 1 h. The reaction flask was
cooled to -20.degree. C. and 115 ml of 10% aqueous sodium hydroxide
was slowly added. During the addition the temperature of the
reaction mixture was maintained at 0.degree. C. and was stirred
vigorously. The mixture was transferred into a 21 separatory
funnel. The remaining residue in the flask was transferred into the
separatory funnel with the aid of a mixture of 5 ml of 10% sodium
hydroxide and 20 ml of dichloromethane. The mixture in the
separatory funnel, which contained some gummy yellow semi-solid,
was shaken vigorously for 10 minutes and the aqueous layer was
allowed to separate slowly from the organic phase. The organic
layer was discarded and the aqueous phase was washed with 100 ml of
dichloromethane and was cooled to 0.degree. C. To the aqueous phase
was added 12.5 ml of concentrated hydrochloric acid (HCl). This pH
of this solution was adjusted to pH 10 with concentrated ammonium
hydroxide. The resulting mixture was extracted with 12.times.250 ml
of chloroform, dried with anhydrous sodium sulphate, concentrated
and yielded 3.1 g of 6-hydroxycinchonine (1). The mother aqueous
layer was extracted with 2.times.200 ml of n-butanol and yielded an
additional 1.0 g to provide a total yield of 4.1 g of
6-hydroxycinchonine (1) (13.2 mmol, 86%). MS, IR and NMR data
confirmed the identity of the compound.
Example 2
Preparation of
(9S)-4-[(9-Hydroxycinchonan-6'yl)oxy]butanenitrile(2).
To a solution of 400 mg (1.2 mmol) of 6-hydroxycinchonane (1) in 25
ml of anhydrous acetone (dried and distilled over potassium
carbonate) and 5 ml of anhydrous dimethylformamide was added 267 mg
(1.93 mmol) of anhydrous potassium carbonate followed by 129 ml
(0.87 mmol) of 4-bromobutyronitrile and a catalytic amount (3 mg)
of 18-crown-6. The reaction mixture was heated to reflux for 18 h,
cooled and filtered. The filtrate was concentrated under reduced
pressure and redissolved in 200 ml of chloroform. The organic layer
was washed with 2.times.50 ml of 5% aqueous sodium hydroxide,
washed with brine, dried using anhydrous magnesium sulfate and
yielded 310 mg (0.82 mmol, 66%) of
(9S)-4-[(9-hydroxycinchonan-6'-yl)oxy]butanenitrile (2) as pale
yellow solids. MS, IR and NMR data confirmed the identity of the
compound.
Example 3
Preparation of (9S)-4-[(9-hydroxycinchonan-6'-yl)butanimidic acid
methyl ester (3).
Hydrochloric acid gas was bubbled through a solution of 200 mg
(0.52 mmol) of (9S)-4-[(9-hydroxycinchonan-6'-yl)oxy]butanenitrile
(2) in 5 ml of anhydrous methanol at -10.degree. C. for a period of
15 minutes. The reaction mixture was stoppered and left at
4.degree. C. for 4 days, concentrated to dryness and yielded 220 mg
(0.49 mmol, 93%) of (9S)-4-[(9-hydroxycinchonan-6'-yl)butanimidic
acid methyl ester (3) as solids. HNMR indicated the purity of
(9S)-4-[(9-Hydroxycinchonan-6'-yl)butanimidic acid methyl ester (3)
was 75% with the remaining 25% as the hydrolyzed compound.
Example 4
Preparation of 9(S)-6'-Cyanomethyloxy cinchonane-9-ol (wherein
L=1).
To a solution of 0.5 g (1.59 mmole) of 6'-hydroxycinchonine and 5
ml of dry DMSO is added slowly over a period of 0.5 h 2.0 ml of
n-butyl lithium (1.6M/hexane, Aldrich). The reaction flask is
cooled in an ice bath and then treated with 96 mg (1.27 mmole) of
chloroacetonitrile. The temperature of the reaction mixture is
allowed to rise to room temperature over a period of 2 h and the
mixture is stirred for 1 h at room temperature. The reaction
mixture is poured into 25 ml of deionized water and 5 ml of ethyl
acetate during which some of the product oils out. 100 ml of
dichloromethane is added to dissolve the oily product and produce
two layers. The heterogeneous brown mixture is concentrated under
reduced pressure to remove the organic solvents during which the
product precipitates. The mixture is left to stand overnight in the
refrigerator. The precipitate is collected and washed with ETOAc to
yield an off-white solid, 0.4 g.
Example 5
Preparation of 6'-Cyanoethyloxy cinchonine (wherein L=2).
To a solution of 0.5 g (1.59 mmol) of 6-hydroxycinchonine in 25 ml
of anhydrous acetone (dried and distilled over potassium carbonate)
and 5 ml of anhydrous dimethylformamide is added 0.329 g (2.39
mmol) of anhydrous potassium carbonate followed by 0.142 g (1.59
mmol) of 3-chloropropionitrile (Aldrich) and 3 mg of 18-crown-6.
The reaction mixture is heated to reflux for 18 h, cooled and
filtered. The filtrate is concentrated under reduced pressure and
redissolved in 200 ml of chloroform. The organic layer is washed
with 2.times.50ml of 5% aqueous sodium hydroxide, washed with brine
and dried using anhydrous magnesium sulphate to yield 0.415 g (1.13
mmol, 71%) of 6'-cyanoethyloxy cinchonine as pale yellow
solids.
Example 6
(9S)-6'-(3-Cyanobenzyloxy) cinchonane-9-ol (wherein L=benzyl).
To a solution of 0.6 g (1.91 mmol) of 6-hydroxycinchonine in 25 ml
of anhydrous acetone (dried and distilled over potassium carbonate)
and 8 ml of anhydrous dimethylformamide is added 0.516 g (2.87
mmol) of anhydrous potassium carbonate followed by 0.329 g (1.91
mmol) of 3-(bromomethyl)benzonitrile (Lancaster) and 4 mg of
18-crown-6. The reaction mixture is heated to reflux for 18 h,
cooled and filtered. The filtrate is concentrated under reduced
pressure and redissolved in 200 ml of chloroform. The organic layer
is washed with 2.times.50 ml of 5% aqueous sodium hydroxide, washed
with brine, dried with anhydrous magnesium sulphate to yield 0.647
g (1.51 mmol, 79%) of (9S)-6'-(3-cyanobenzyloxy) cinchonane-9-ol as
pale off-white solids.
Example 7
Preparation of quinidine immunogen
(9S)-4-[(9-hydroxycinchonan-6'yl)oxy]-1-iminobutyl-[BTG] (4).
A freshly prepared solution of 166 mg of compound
(9S)-4-[(9-hydroxycinchonan-6'-yl)butanimidic acid methyl ester (3)
in 1 ml of dry DMSO was added rapidly to a solution mixture of 55.5
ml of DMSO and 0.1M potassium phosphate (KPi) pH 7.5 (3:1)
containing 700 mg of bovine thyroglobulin (BTG). The reaction
mixture was stirred overnight at 4.degree. C. The resulting
conjugate was placed in a dialysis tube (50,000 MW cut-off) and was
dialyzed in a 3:1 mixture of DMSO/50 mM KPi pH 7.5, 1:1 mixture of
DMSO/50 mM KPi pH 7.5, 1:3 mixture of DMSO/50 mM KPi pH 7.5, and
twice in 50 mM KPi buffer pH 7.5. The conjugate was removed and
sterile filtered to yield 110 ml solution of 6.5 mg/ml as
determined by protein assay (Coomasie Blue). The degree of lysine
modification of this conjugate was determined by the ability of the
remaining lysine residues to react with trinitrobenzenesulfonic
acid (TNBS). The resulting yellow complex was then measured at 420
nm. The results indicated that 63% of the available lysines in the
quinidine immunogen had been modified. This material was used for
animal immunization.
Example 8
Preparation of quinidine-bovine serum albumin (BSA) conjugate
(9S)-4-[(9-hydroxycinchonan-6'yl)oxy]-1-iminobutyl-[BSA].
To a solution of BSA (1.2 g in 19 ml of 0.1M (KPi) pH 7.5)
containing 57 ml of DMSO was added rapidly a freshly prepared
solution of (9S)-4-[(9-hydroxycinchonan-6'-yl)butanimidic acid
methyl ester (3) (23 mg in 1 ml of dry DMSO). The reaction was
stirred overnight at 4.degree. C. The resulting conjugate was
placed in a dialysis tube (10,000 MW cut-off) and was dialyzed in a
3:1 mixture of DMSO/50 mM KPi pH 7.5, 1:1 mixture of DMSO/50 mM KPi
pH 7.5, 1:3 mixture of DMSO/50 mM KPi pH 7.5, and twice in 50 mM
KPi buffer pH 7.5. The conjugate was removed, sterile filtered and
yielded a 115 ml solution of 10.2 mg/ml as determined by protein
assay (Coomasie Blue). This material served as the
conjugate-capture for the antibody screening by ELISA.
Example 9
Preparation of disubstituted quinidine derivative
(S)-8-ethenyl-2-[hydroxy-6-(2-ethoxy-2-oxoethoxy)-4-quinolinylmethyl]-1-(2
-ethoxy-2-oxoethyl)azoniabicyclo[2.2.2]octane iodide (15).
A 1-l 3-necked, round-bottomed flask equipped with a magnetic
stirrer was charged with 2.0 g (6.44 mol) of 6-hydroxycinchonine
(1) and 10 ml of DMSO which had been dried over molecular sieves.
To the stirred solution was added very slowly over a period of 0.5
h 2.8 ml (7.0 mmol) of n-butyl lithium (2.5M/hexane, Aldrich). The
reaction flask was cooled with an ice-water bath and then treated
with 1.6 ml (13.57 mmol) of ethyl iodoacetate. The temperature was
allowed to rise to room temperature over a period of 2 h and the
mixture was stirred at room temperature for 1 h. The reaction
mixture was poured into 65 ml of deionized water and 15 ml of ethyl
acetate during which some of the product oiled out. 100 ml of
dichloromethane was added to dissolve the oily product and produce
two layers. The heterogeneous brown mixture was concentrated under
reduced pressure to remove the organic solvents during which the
product precipitated. The mixture was left to stand overnight in
the refrigerator. The precipitate was collected and washed with
ETOAc and yielded 1.6 g (41%) of
(S)-8-ethenyl-2-[hydroxy-6-(2-ethoxy-2-oxoethoxy)-4-quinolinylmethyl]-1-(2
-ethoxy-2-oxoethyl)azoniabicyclo[2.2.2]octane iodide as an
off-white solid. NMR, IR and MS data confirmed compound
identity.
Example 10
Preparation of disubstituted quinidine derivative
(9S)-1-(3-carboxypropyl)-6'-(3-carboxypropoxy) cinchoninum chloride
(16).
To a solution of 2.7 g (8.7 mmol) of (1) in 75 ml of dry acetone
(dried and distilled over potassium carbonate) and 25 ml of
dimethylformamide (Aldrich, 99%) was added 2.5 g (18.1 mmol) of
anhydrous potassium carbonate, followed by 2.49 ml (17.4 mmol), of
ethyl-4-bromobutyrate and 25 mg of 18-crown 6. The reaction mixture
was heated under reflux for 18 h, cooled, filtered and the filtrate
was concentrated under reduced pressure. The residue was purified
by silica gel column chromatography using a mixture of 6:1:1:1
ethyl acetate:methanol:water:acetone as the eluent to give 2.7 g of
diethylester as a brown oil. This oil was treated with 1.4 g of
potassium carbonate in 150 ml of methanol and heated to reflux for
20 h. The reaction mixture was concentrated and redissolved in 15
ml of water. The resulting solution was adjusted to pH 5 with 1N
HCl. This was concentrated under reduced pressured and the brown
residue was purified on preparative thin layer chromatography using
a mixture of 8:1:1 ethyl acetate:methanol:water and yielded 2.34 g
(51%) of diacid as off-white powders. MS, IR and NMR data confirmed
the identity of the compound
Example 11
Preparation of (9S)-4-[(9-Hydroxycinchonan-6'-yl) oxybutanoic acid
ethyl ester (5).
To a solution of 250 mg (0.80 mmol) of (1) in 6 ml of dry
dimethylformamide (Aldrich, 99%) was added 110 mg (0.80 mmol) of
anhydrous potassium carbonate, followed by 109 ml (0.75 mmol) of
ethyl-4-bromobutyrate and catalytic amount (4 mg) of 18-crown 6.
The reaction mixture was heated at 120.degree. C. for 2 h, cooled,
and placed under reduced pressure to remove dimethylformamide. 50
ml of dichloromethane was added to the residue and the mixture was
filtered. The filtrate was washed with 2.times.25 ml of 5% sodium
hydroxide, brine, dried (anhydrous sodium sulphate) and
concentrated. The residue was purified on preparative thin layer
chromatography using a mixture of 6:1:1:1 ethyl
acetate:methanol:water:concentrated ammonium hydroxide as the
eluent and yielded 160 mg (0.37 mmol, 45%) of (5) as brown oil. MS,
IR and NMR data confirmed the identity of the compound.
Example 12
Preparation of (9S)-4-[9-Hydroxycinchonan-6'-yl)oxybutanoic
acid(6).
A mixture of 150 mg (0.35 mmol) of (5) and 80 mg of potassium
carbonate in 35 ml of methanol was heated to reflux for 20 h. The
reaction mixture was concentrated and redissolved in 15 ml of
water. The resulting solution was adjusted to pH 5 with the
addition of 1N HCl. This was concentrated under reduced pressure
and the brown residue was purified on preparative thin layer
chromatography using a mixture of 8:1:1 ethyl
acetate:methanol:water and yielded 85 mg (0.21 mol, 61%) of (6) as
off-white powders. MS, IR and NMR data confirmed compound
identity.
Example 13
Preparation of
(9S)-N-[(3',6'-dihydroxy-3-oxospiro[isobenzofuran-1(3H),9'-[9H]xanthen]-5y
l) methyl]-4-[(9-hydroxycinchonan-6'-yl)oxy]butamide (7).
To 50 mg (0.12 mmol) of (6) was added 5 ml of dimethylformamide.
After cooling to 0.degree. C., to the resulting solution was added
30 mg (0.14 mmol) of dicyclohexylcarbodiimide and 25 mg (0.21 mmol)
of N-hydroxysuccinimide. The mixture was allowed to stir at
4.degree. C. for 48 h and set aside. In another flask was added 60
mg (0.15 mmol) of 5-aminomethyl-fluorescein, hydrochloride and 3 ml
of pyridine at room temperature. The precipitate of pyridine
hydrochloride immediately appeared. To this suspension was added
dropwise the previously prepared N-hydroxysuccinimide solution. The
mixture was allowed to stir at room temperature for 4 days and then
was concentrated under reduced pressure. The residue was purified
on preparative thin layer chromatography (silica, 2 mm) using a
mixture of 9:1 ethyl acetate:methanol as the eluent. The orange
product obtained indicated the presence of impurities and was
repurified on thin layer chromatography (silica, 0.25 mm) using a
mixture of 5:1 chloroform:methanol and yielded 24 mg (0.032 mmol,
26%) of (7). MS, IR and NMR data confirmed compound identity.
Example 14
Preparation of
(9S)-[[[(6'-methoxycinchonan-9-yl)oxy]carbonyl]amino]acetic acid
ethyl ester (8).
To a solution of 200 mg (0.61 mmol) of quinidine in 5 ml of dry
dichloromethane was added 74 ml (0.65 mmol) of
ethylisocyanatoacetate. The mixture was allowed to stir
magnetically at room temperature for 18 h and concentrated under
reduced pressure. The residue was purified on preparative thin
layer chromatography (silica, 2 mm) using a mixture of 1:9 ethyl
acetate:methanol as the eluent and yielded 130 mg (0.28 mmol, 46%)
of (8). MS, IR and NMR data confirmed identity of the compound.
Example 15
Preparation of (9S)-[[[(6'-methoxycinchonan-9-yl)
oxy]carbonyl]amino]acetic acid (9).
A solution of 120 mg (0.26 mmol) of (8) in 2 ml of methanol was
added 200 mg of potassium carbonate and 0.5 ml of water. The
mixture was heated to reflux for 2 h and cooled to room
temperature. The reaction mixture was filtered and the filtrate was
concentrated to remove methanol. The residue was redissolved in 2
ml of water and 1N HCl was added dropwise to the solution until the
pH reached 7. The aqueous solution was concentrated, 100 ml of
methanol was added and filtered. The filtrate was concentrated and
yielded 95 mg (0.22 mmol, 85%) of (9).
Example 16
Preparation of
(9S)-[2-[[(3',6'-dihydroxy-3-oxospiro[isobenzofuran-1(3H),9'-[9H]pantheon]
-5-yl) methylamino]-2-oxoethyl] carbamic acid
6'-methoxycinchonan-9-yl ester (10).
A solution of 26 mg (0.061 mmol) of (9) in 0.5 ml of
dimethylformamide was cooled to 0.degree. C. To the solution was
added 20 mg (0.096 mmol) of DCC and 16 mg (0.0139 mmol) of
N-hydroxysuccinimide. The reaction mixture was stirred at 4.degree.
C. for 20 h and set aside. In another flask was added 30 mg (0.075
mmol) of 5-aminomethylfluorescein hydrochloride and 3 ml of
anhydrous pyridine. A precipitate of pyridine hydrochloride formed.
To this suspension was added dropwise the previously prepared (in
situ) solution of N-hydroxysuccinimide ester. The reaction mixture
was allowed to stir at room temperature for 48 h and concentrated
under reduced pressure. The residue was applied to preparative thin
layer chromatography (silica, 2 mm). The plates were developed
using a mixture of 8:2 chloroform:methanol as the eluent. The
yellow product obtained was repurified using the above eluent and
yielded 11 mg (0.014 mmol, 24%) of (10) as orange solids. MS, IR
and NMR data confirmed the identity of the compound.
Example 17
Preparation of
(9S)-2-[3-[(9-hydroxycinchonan-6'-yl)oxy]propyl]-1H-isoindole-1,3(2H)-dion
e (11).
A mixture of 6-hydroxycinchonine (1) (2.5 g, 8.06 mmol),
N-(3-bromopropyl)phthalimide (2.16 g, 8.06 mmol), anhydrous
potassium carbonate (1.3 g, 9.4 mmol) and 18-crown-6 (5 mg) in 75
ml of dry acetone was allowed to reflux for 16 h under argon
atmosphere. The reaction was cooled to room temperature and 30 ml
of methanol was added to make a homogeneous solution. The resulting
solution was concentrated and the residue was partially purified on
silica gel column chromatography using 5% methanol in chloroform as
the eluent. This product was repurified on silica gel column
chromatography using 10% methanol in chloroform and yielded 1.08 g
(2.17 mmol, 27%) of (11) as off-white solids. MS, IR and NMR data
confirmed compound identity.
Example 18
Preparation of (9S)-6'-(3-aminopropoxy) cinchoninan-9-ol (12).
To 300 mg (0.60 mmol) of the quinidine propyl phthalimide (11) was
added 2 ml of methyl amine in methanol saturated with gaseous
methyl amine. The reaction flask was stoppered and the mixture was
allowed to stir at room temperature for 16 h. An aliquot of the
reaction mixture was monitored by thin layer chromatography (10%
methanol in dichloromethane) and indicated the complete
disappearance of starting material. The reaction mixture was
concentrated and the residue was redissolved in 30 ml of
dichloromethane. The organic layer was extracted twice with an
equal volume of water. The aqueous portion was concentrated and
yielded 124 mg (0.33 mmol, 56%) of (12) as a clear oil. MS, IR and
NMR data confirmed compound identity.
Example 19
Preparation of
(9S)-3',6'-dihydroxy-N-[3-[(9-hydroxycinchonan-6'-yl)
oxy]propyl]-3-oxospiro[isobenzofuran-1 (3H),
9'-[9H]xanthene]-5-carboxamide(13).
A mixture of 36 mg (0.097 mmol) of the quinidine propylamine (12)
and 5-carboxyfluorescein N-hydroxysuccinimide ester (37 mg, 0.098
mmol) in 2.5 ml of dry pyridine was allowed to stir magnetically at
room temperature for 3 days under argon atmosphere. The reaction
was monitored by thin layer chromatography (8:1:1 ethyl
acetate:methanol:water), which indicated the presence of
substantial quantities of starting materials. This was then heated
between 40.degree.-45.degree. C. for 3 days under argon atmosphere.
The mixture was cooled to room temperature and concentrated under
reduced pressure. The residue was purified on silica gel column
chromatography using 8:1:1 ethyl acetate:methanol:water as the
eluent and yielded 4 mg (5.5.times.10-3 mmol, 5.6%) of (13) as
orange solids. MS, IR and NMR data confirmed compound identity.
Example 20
Production of polyclonal antibody P796.
For production of polyclonal antibody, a sheep and goat were
injected with 1 mg each of the immunogen. The first immunization,
using complete Freund's Adjuvant, consisted of multiple injections
carried out across the back of the animals. After one week, the
second immunization containing 1 mg of the immunogen and incomplete
Freund's Adjuvant was injected. Injections were repeated at the
third and fourth weeks. Thereafter, the animal received a monthly
injection of 3 mg of the immunogen. After 6 months the animal was
bled, the blood was allowed to clot, the clot was centrifuged at
about 3000 rpm for 15-20 minutes and the serum was separated by
means of decantation.
Example 21
Production of monoclonal antibody MoAb Q6-6C5.
For production of the monoclonal antibody, the immunogen for
quinidine was injected into female Balb/c mice. The first
immunization contained complete Freund's Adjuvant and the second
immunization contained incomplete Freund's Adjuvant. Spleen cells
from the immunized mouse were fused with NSO myeloma cells in a
ratio of 4:1 in the presence of polyethylene glycol (PEG) according
to a modification procedure of Kohler and Milstein, Nature, 256
495-497(1975). Hydridoma cell culture supernatants containing
monoclonal antibody (MoAb) were screened by ELISA method using BSA
coated plates and detected with rabbit anti-mouse Ig conjugated to
alkaline phosphatase. The final selection of monoclonal antibody
Q6-6C5 was achieved by ELISA followed by analysis in the FPIA assay
system.
Example 22
Fluorescence polarization immunoassay (FPIA).
The following assay reagents and protocol were used on the
automated COBAS FARA II.RTM. analyzer configured for fluorescence
polarization determinations for quinidine which are given in this
application. (Roche Diagnostic Systems Inc., Somerville, N.J., A
subsidiary of Hoffmann-La Roche Inc., Nutley, N.J.).
I. Reagent Formulation for Monoclonal Assay:
a. Tracer reagent.
50 mM ACES (N-2-acetamido-2-aminoehanesulfonic acid), pH6.5
0.01% (w/v) bovine gamma globulin
0.09% (w/v) sodium azide
Tracer concentration: 6.times.10-7M
b. Monoclonal antibody reagent.
0.1M phosphate, pH7.5
150 mM sodium chloride
0.09% sodium azide
0.05% bovine serum albumin
Antibody dilution: 1:550 in antibody buffer.
c. Quinidine calibrator
0, 0.5, 1, 2, 4, and 8 ug/mL of quinidine in treated normal human
serum, 5 mM EDTA with 0.09% sodium azide.
d. Sample diluent: Cobas FP Sample Dilution Reagent, code 44268
II. Assay protocol
Mixed 2.6 .mu.l sample with 23.4 .mu.l sample diluent. Added 200
.mu.l antibody reagent and read background. Added 30 .mu.l tracer
reagent. Incubated for 30 sec. Read fluorescence polarization at
520 nm.
* * * * *